Expression and immunoaffinity purification of recombinant soluble human GPR56 protein for the analysis of GPR56 receptor shedding by ELISA

Tai Yun Yang, Nien Yi Chiang, Wen Yi Tseng, Hsiao Lin Pan, Yen Ming Peng, Jiann Jong Shen, Kuo An Wu, Ming Ling Kuo, Gin Wen Chang, Hsi Hsien Lin*

*Corresponding author for this work

Research output: Contribution to journalJournal Article peer-review

9 Scopus citations

Abstract

GPR56 is a multi-functional adhesion-class G protein-coupled receptor involved in biological systems as diverse as brain development, male gonad development, myoblast fusion, hematopoietic stem cell maintenance, tumor growth and metastasis, and immune-regulation. Ectodomain shedding of human GPR56 receptor has been demonstrated previously, however the quantitative detection of GPR56 receptor shedding has not been investigated fully due to the lack of appropriate assays. Herein, an efficient system of expression and immune-affinity purification of the recombinant soluble extracellular domain of human GPR56 (sGPR56) protein from a stably transduced human melanoma cell line was established. The identity and functionality of the recombinant human sGPR56 protein were verified by Western blotting and mass spectrometry, and ligand-binding assays, respectively. Combined with the use of two recently generated anti-GPR56 monoclonal antibodies, a sensitive sandwich ELISA assay was successfully developed for the quantitative detection of human sGPR56 molecule. We found that GPR56 receptor shedding occurred constitutively and was further increased in activated human melanoma cells expressing endogenous GPR56. In conclusion, we report herein an efficient system for the production and purification of human sGPR56 protein for the establishment of a quantitative ELISA analysis of GPR56 receptor shedding.

Original languageEnglish
Pages (from-to)85-92
Number of pages8
JournalProtein Expression and Purification
Volume109
DOIs
StatePublished - 05 2015

Bibliographical note

Publisher Copyright:
© 2014 Elsevier Inc. All rights reserved.

Keywords

  • Adhesion-class G protein-coupled receptor
  • ELISA
  • GPR56
  • Soluble GPR56

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